PROJECT SUMMARY/ABSTRACT Distinct pathogenic mechanisms lead to airway obstruction, morbidity, and mortality in different subsets of individuals with asthma. In about half of individuals with asthma, the type 2 cytokine IL-13 acts directly on airway epithelial cells to induce secretion of the airway mucin MUC5AC, which becomes tethered to the airway epithelium leading to airway obstruction. There is an urgent need to understand this process better since some individuals with type 2 asthma have severe disease that responds poorly to available therapies. Standard therapies against type 2 inflammation are also often ineffective in the many individuals with type 2-low asthma. The long-term goal of this project is to discover new epithelial cell mechanisms and pathways that are important for type 2 and non-type 2 pathophysiology and may be promising targets for therapy of severe asthma. Preliminary data suggest the hypothesis that type 2 asthma susceptibility and severity relate to heightened IL-13-driven epithelial production of MUC5AC and to dramatic changes in airway epithelium that alter mucus gel organization and physical properties. In addition, we have new evidence that a subset of individuals with asthma have interferon (IFN)-driven inflammation and that the IRE1 ER stress pathway contributes to pathogenesis in both type 2 and IFN-driven inflammation. We will address these hypotheses in three specific aims. Specific aim 1 will examine how differences in airway epithelial cell responses to IL-13 contribute to type 2-high asthma susceptibility and severity. Using cultures of primary human bronchial epithelial (HBE) cells from individuals with asthma and healthy controls, we will carefully measure inter- individual differences in MUC5AC induction by IL-13, identify the regulatory pathways that underlie these differences, and relate these differences to disease status and severity. Specific aim 2 will identify changes in airway epithelial secretory cells and mucus that lead to mucostasis and airway obstruction in asthma. Here we will test the hypotheses that IL-13 induces changes in the macro- and micro-rheologic properties of mucus that are specific to type 2 high asthma. We will also determine how IL-13-induced changes in mucin cross-linking, mucin glycosylation state, and protease/anti-protease activity contribute to altered rheologic properties, tethering, and mucostasis. Specific aim 3 will define an HBE cell ER stress signature by inducing ER stress in cultured HBE cells and determine how inhibiting the IRE1 ER stress pathway affects HBE cell responses to HDM, IL-13, and IFN. These studies, together with highly related studies in human subjects and mouse models from Project 2 and the Clinical Subject and Biospecimen Core, will have a substantial impact by identifying new mechanisms that contribute to type 2 asthma susceptibility and severity and by testing the therapeutic potential of targeting the IRE1-mediated ER stress response to meet unmet therapeutic needs in individuals with asthma refractory to current therapies.